Folate cofactors, in particular members of the B vitamin complex, play an important role in cellular metabolism and growth by serving as coenzymes for the synthesis of purines and pyrimidines, the precursors of RNA and DNA. Plasmodium spp. and other microorganisms synthesize folate cofactors by using guanosine triphosphate (GTP) as a precursor. Plasmodium spp. can also salvage folate cofactors from the mammalian host. Two important enzymes in the folate biosynthesis pathway, namely dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), are targets of sulfa drugs (anti-DHPS) and anti-DHFR antifolates (pyrimethamine, cycloguanil, and WR99210) respectively. Species inhibited by these drugs include parasitic protozoa such as Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii and Isospora belli, and bacteria such as Haemophilus influenzae, Escherichia coli and Klebsiella pneumonia. Anti-DHPS drugs and anti-DHFR drugs are synergistic. Using both types of drugs in combination gives much enhanced efficacy, and consequently can extend the clinically effective lifetime of the drugs when compared with using either drug alone. The advantage of using combination drugs is that they can forestall the rise of drug resistance. In antifolate-resistant Plasmodium falciparum, a number of drug-resistance mutations have been found, including five mutations in dihydropteroate synthase and four mutations in dihydrofolate reductase. Although resistant mutations are present, the two drug targets are still valid for new drug development, with the provision that the new compounds must effectively inhibit both drug sensitive and resistant variants.
In order to evaluate antimalarial activity of compounds, in vitro antimalarial screening assay using malaria parasites grown in human red blood cells has been widely employed. However, this assay is labor intensive, expensive, and requires specialized personnel and equipment. The parasites are cultivated in red blood cells with culture media containing human serum or a suitable substitute. Routine changing of culture media and supply of new blood cells for the parasites are needed. Moreover, evaluation of drug efficacy requires microscopic, fluorescent, or radioactive methods for measuring parasite growth. These requirements are a hindrance to high throughput screening and limit antimalarial screening to laboratories with malaria culture systems in place. For target-based anti-malarial screening (which includes antifolates), surrogate models are useful alternatives when malaria culture facilities are not available. In Plasmodium spp., a single gene encodes a protein with DHFR and thymidylate synthase (TS) activities, making a bifunctional enzyme DHFR-TS, while in bacteria, these enzymes are encoded by separate genes, folA and thyA. Similarly, DHPS exists as a bifunctional enzyme with 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK) in Plasmodium spp. However, in bacteria, these proteins are encoded by folK and folP genes, respectively. A bacterial surrogate model screening system can be established using a cell line defective for an essential bacterial gene(s) function such that the cell can grow only in prototrophic medium with the essential factor provided exogenously. The bacterial surrogate transformed with a complementary gene (e.g. homologous gene from parasite species) is able to grow in auxotrophic medium. In the presence of compounds that are inhibitory to the complementing gene, the growth of the surrogate is inhibited in auxotrophic medium. In general, growth inhibition of the bacterial surrogate correlates well with growth inhibition of the pathogenic parasite species from which the complementing gene was derived from.
Although previous bacterial surrogate models for screening compounds with parasite DHFR and DHPS inhibitory activities have been described, none has the capacity to test both types of drugs at the same time. This is because other anti-bacterial drugs are required to establish the knockout of bacterial host gene activities to make the cell dependent on parasite gene activity for growth. For bacterial surrogate models for testing anti-PfDHFR activity, endogenous bacterial DHFR was inactivated either by adding trimethoprim (chemical inhibitor of E. coli's DHFR) (Bunyarataphan S., et al. 2006, Chusacultanachai S. et al. 2002), or using the PA414 strain, a folA knockout strain (Ahrweiler P. M., et al 1988, Prapunwattan P., et al 1996) or using DHFR deleted Saccharomyces cerevisiae mutant (Wooden J. M., et al. 1997, Djapa L. Y., et al. 2007). The anti-parasitic activities for some compounds may not correlate well with the results from the aforementioned DHFR surrogate models, since only the DHFR encoding gene is complemented. These surrogates thus do not truly model parasite DHFR function, which exists as a bifunctional enzyme with TS (see above). For DHPS surrogate models, folP deficient E. coli, C600 strain has been used as a bacterial surrogate model (Fermer C., et al 1997, Berglez J., et al. 2004). However, all of these bacterial surrogates have poor growth rates since they are derived from E. coli K strains. In some case, E. coli BL21(DE3) strain has been mutated to be folA and thyA deficient, but still contain antibiotic selectable markers, which are left behind as a result of the process to disrupt the bacterial gene (Sinekiewicz N., et al. 2008). Indeed, all of the above mentioned surrogate models retain antibiotic selectable markers. The retention of these markers is also disadvantageous for screening anti-parasitic drug combinations against multiple targets. This is because it not only limits introduction of multiple plasmids, or other recombinant DNA vectors carrying complementing parasite genes. For example, two different antibiotic markers would be needed to co-transform two plasmids, one with cloned parasite DHFR-TS gene complementing folA and thyA, and another one with cloned parasite HPPK-DHPS gene complementing folK and folP. The standard procedures for generating gene knockouts in E. coli typically employ kanamycin and/or chloramphenicol resistance markers, which are also commonly-used markers for transformation vectors. Whilst it is facile to transform say, a surrogate cell harboring a single kanamycin resistance marker with a plasmid bearing a beta-lactamase marker, it becomes increasingly difficult to find suitable antibiotic resistance markers for plasmid transformation as the number of genes knocked out in the surrogate increases. Unless a strategy is employed to remove resistance markers, no suitable markers would be available for selection of cells transformed with complementing genes as the surrogate would already be resistant to multiple different antibiotics. For this reason, there is currently no surrogate screening system for testing combination drugs against DHFR-TS and HPPK-DHPS. Drug combinations against these targets are important, as they have been proven to be effective anti-parasitic chemotherapies which is used to treat malaria.
In the subject invention, a model is described for testing combination drugs which inhibit both parasite DHFR-TS and HPPK-DHPS enzyme activities. This invention entails an Escherichia coli strain whose thyA, folA, folK and folP genes were sequentially disrupted using genetic knockout coupled with elimination of antibiotic resistance markers. We evaluated the use of this invention as a host for screening inhibitors against DHFR and/or DHPS of Plasmodium or other apicomplexan parasites. The tools, thyAfolA knockout (KO), folKfolP KO and thyAfolAfolKfolP KO E. coli are easy and convenient for testing single and combination drugs as plasmids bearing complementing parasite genes can be introduced simply by transformation. The assay results using this model are consistent with those from the conventional antimalarial screening method using malaria parasite. With this tool, it is feasible to perform antifolate screening against malaria and other parasitic disease in a laboratory with facilities for bacteria cell culture, which more are widely available than parasite culture facilities.